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Application Notes

Response to Drugs–Inside and Outside of 3D Cell Cultures

The effect of drugs on 3D cell cultures was evaluated through cell imaging.
The differences in the effect of drugs on the outside and inside of 3D cell cultures such as spheroids was investigated.


Recently, 3D cell cultures, such as spheroids, were used to study the response of cells to drugs. However, the difference in response on the inside and outside of 3D cell cultures has not been considered. In this study, this difference was investigated through the observation at depth of spheroids with a confocal microscope.


Preparation of samples

Fucci is a cell-cycle indicator using a dual color scheme. Spheroids composed of Fucci-expressing transgenic HT- 29 cells were exposed to 10 nM of cisplatin. After incubating for 96 hours, the medium containing cisplatin was removed from the cell cultures, and they were fixed in 4% formalin and treated with transparentizing reagent.

Acquisition and analysis of fluorescent images

Confocal fluorescent images of the above-mentioned spheroids were obtained at every 5 μm thickness increment, from the surface to 150 μm in depth. The luminosity ratio of red (G0 and G1 phases) to green (S, G2, and M phases) in each layer was analyzed with analysis software. In the absence of cisplatin, there was no significant difference in the red/green ratio between outside and inside (Figure 1). In the presence of 10 nM of the cisplatin, the red/green ratio increased with an increase in distance from the surface (Figures 2 and 3).


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